DESCRIPTION: (Scanned from the applicant's description) Type 2 diabetes mellitus is characterized by chronic hyperglycemia and is often associated with elevated plasma lipid levels. The overall objective of this proposal is to ascertain the mechanisms whereby prolonged exposure to elevated levels of fatty acids (FA) affects pancreatic beta-cell function in Type 2 diabetes. Previously, we have demonstrated that prolonged exposure to FA impairs insulin gene expression only in the presence of high glucose, and that this is associated with increased neutral lipid synthesis. Specific Aim I: To identify the metabolic intermediate(s) generated along the pathway of neutral lipid synthesis responsible for the impairment of insulin secretion and gene expression upon prolonged exposure to FA. Isolated rat islets, HIT-T15, and betaHC-l3 cells will be cultured for 1 to 7 days in the presence of increasing concentrations of glucose and FA. Pharmacological tools will be used to inhibit or stimulate each step of neutral lipid synthesis, in order to identify the metabolic intermediate(s) generated along the esterification pathway (i.e., long-chain Acyl-CoA, diacyiglycerols, or triacylglycerols) responsible for the FA-induced impairment of beta-cell function. Specific Aim II: To assess whether the glucose-dependent deleterious effects of prolonged exposure to elevated FA on beta-cell function are glucose-specific, and whether the mechanisms of these effects are transcriptional, post-transcriptional, or translational. beta-cell exhaustion will be distinguished from bona fide toxicity in experiments where diazoxide will be used to inhibit insulin release. The glucose-specificity of PA effects will be investigated by using a non-glucose secretagogue to stimulate insulin secretion and insulin gene expression. The glucose-dependent effects of FA on proinsulin biosynthesis, insulin mRNA stability, and endogenous insulin gene transcription will be assessed. The effects of FA on insulin promoter activity will be characterized in HIT-Tl5 and betaHC-13 cells and also investigated in primary islets using the recombinant adenovirus system. Specific Aim III: To determine whether high-fat feeding in hyperglycemic Goto-Kakizaki (GK) rats impairs insulin secretion, insulin biosynthesis, and insulin gene expression, and whether these effects are prevented by normalization of blood glucose levels. GK or control rats will be fed a high-fat diet for 6 weeks, after which insulin secretion, proinsulin biosynthesis, and insulin gene expression will be assessed. Blood glucose levels will be normalized in GK rats by phloridzin administration, in an attempt to prevent the deleterious effects of high-fat diet on beta-cell function. These experiments will provide important insights into the pathophysiology of beta-cell dysfunction of type 2 diabetes, and have clear implications for the treatment of this disease.